Mice play a crucial role as the prime model organism to study many aspects of development and function in hematology and immunology. However, their habitats and pathogens that shape and constantly challenge the immune system have diverged between species, resulting in the fact that genes related to immunity, together with genes involved in reproduction and olfaction, are the most divergent between the two species (2004, Mestas and Hughes, J Immunol 172:2731-2738). Mice rendered genetically suitable to support human cells and tissues have become a favorite model bridging the gap between mouse models and studies in humans (2009, Legrand et al., Cell Host Microbe 6:5-9; 2007, Shultz et al., Nat Rev Immunol 7:118-130; 2007, Manz, Immunity 26:537-541). Particularly, mice that reconstitute a functional human immune system after engraftment of hematopoietic stem and progenitor cells (HSPCs) are of high interest to study vaccine candidates and the biology of pathogens restricted to humans in vivo. To achieve efficient xenotransplantation, mice lacking an adaptive immune system and natural killer (NK) cells have been successfully developed in the last years and the major models differ mainly in the background strains used. The first one employs the BALB/c Rag2−/−γc−/− (DKO) mice, and neonatal intrahepatic HSPC transfer (2004, Traggiai et al., Science 304:104-107; 2004, Gimeno et al., Blood 104:3886-3893). A second model reconstitutes instead NOD/scid/γc−/− (NSG) mice by i.v. or intrahepatic injection of human HSPCs (2002, Ito et al., Blood 100:3175-3182; 2005, Ishikawa et al., Blood 106:1565-1573; 2005, Shultz et al., J Immunol 174:6477-6489). After transfer into these mice, human HSPCs can develop into most of the hematopoietic lineages and the human chimerism is maintained for several months (2004, Traggiai et al., Science 304:104-107; 2005, Ishikawa et al., Blood 106:1565-1573). Overall the composition of engrafted cells is similar in these models but higher human engraftment levels were obtained in NOD-based models (2010, Brehm et al., Clin Immunol 135:84-98). This advantage is thought to be caused at least partially by a polymorphism in the gene encoding the inhibitory receptor signal regulatory protein alpha (SIRPα) (2007, Takenaka et al., Nat Immunol 8:1313-1323).
SIRPα is a transmembrane protein containing three Ig-like domains in its extracellular region and putative tyrosine phosphorylation sites in its cytoplasmic region (2009, Matozaki et al., Trends Cell Biol 19:72-80). SIRPα is strongly expressed in neurons and in macrophages, dendritic cells, and neutrophils. The ligands of SIRPα are CD47 and surfactant A and surfactant D and their binding to the receptor induces the recruitment of phosphatases SHP-1 and SHP-2 to the plasma membrane. In phagocytic cells, this recruitment negatively regulates phagocytosis upon binding to its ligands (2005, Okazawa et al., J Immunol 174:2004-2011). CD47 is ubiquitously expressed in all cells of the body, including all lineages of hematopoietic cells. The inhibitory signaling via CD47− SIRPα ligation has important consequences in vivo because upon transfer into WT mice, CD47−/− cells are rapidly cleared by splenic red pulp macrophages (2000, Oldenborg et al., Science 288:2051-2054). Subsequently it was recognized that the regulation of CD47 expression plays important functions in such diverse biological processes as cell migration, the regulation of the erythrocyte life span, and HSC circulation (2000, Oldenborg et al., Science 288:2051-2054; 2009, Jaiswal et al., Cell 138:271-285; 2003, Motegi et al., EMBO J 22:2634-2644). Whereas it had been recognized that mouse phagocytes regulate human cell and tissue transplantation into mice (2004, Rozemuller et al., Exp Hematol 32:1118-1125; 1997, Terpstra et al., Leukemia 11:1049-1054; 2005, Andres et al., Transplantation 79:543-549), it has been recently demonstrated that, due to allelic variation, partial engagement of NOD SIRPα but not C57BL6 SIRPα on respective phagocytes by human CD47 leads to decreased phagocytosis of human cells in vitro (2007, Takenaka et al., Nat Immunol 8:1313-1323; 2007, Takizawa and Manz, Nat Immunol 8:1287-1289). Given the above discussed additional residual human engraftment impairment, it was hypothesized that expression of human SIRPα (i.e., hSIRPα or huSIRPα) on mouse macrophages would lead to decreased phagocytosis of human CD47-expressing cells (2000, Oldenborg et al., Science 288:2051-2054; 2001, Blazar et al., J Exp Med 194:541-549; 2007, Wang et al., Blood 109:836-842). Thus, to create an improved platform for future generations of humanized mice, human SIRPα transgenic mice were generated that faithfully express the receptor using F1 129/BALB/c Rag2+/−γcy/− ES cells, which allow straightforward and rapid genetic modifications.
Severely immunocompromised mice lacking T cells, B cells, and NK cells have become widely used hosts for the xenotransplantation of human cells due to their diminished rejection of cells and tissues of human origin (2004, Traggiai et al., Science 304:104-107; 2002, Ito et al., Blood 100:3175-3182; 2005, Ishikawa et al., Blood 106:1565-1573; 2005, Shultz et al., J Immunol 174:6477-6489). However, it has been noted that there are additional strain-specific factors that influence engraftment efficiencies as demonstrated by the incapability of C57Bl6 Rag2−/−γc−/−, in contrast to NOD/Rag1−/−γc−/− mice, to support engraftment of human cells. The importance of murine macrophages in xenorejection had been noted more than 10 y ago, but the mechanisms of xenorecognition were only described recently (2007, Takenaka et al., Nat Immunol 8:1313-1323; 2004, Rozemuller et al., Exp Hematol 32:1118-1125; 1997, Terpstra et al., Leukemia 11:1049-1054). It has been established that binding of CD47 on target cells to SIRPα on macrophages sends a “don't eat me” signal to the phagocyte, i.e., murine CD47−/− cells are rapidly cleared from WT mice (2000, Oldenborg et al., Science 288:2051-2054). In the context of xenotransplantation, the advantage of NOD/scid mice as hosts for human cells compared with CB17/scid or C57Bl6/Rag mice was subsequently suggested to require a specific variant of the polymorphic inhibitory receptor SIRPα (2007, Takenaka et al., Nat Immunol 8:1313-1323). A number of polymorphisms in the extracellular domain of SIRPα enabled SIRPα (NOD) to bind to human CD47, whereas SIRPα (C57Bl6) was unable to bind human CD47 (2007, Takenaka et al., Nat Immunol 8:1313-1323). In vitro assays were further used to characterize the direct effect of SIRPα on human hematopoiesis, but it remained formally unconfirmed whether SIRPα is sufficient for the enhanced engraftment in NOD-based strains. Notably, the NOD strain is characterized by a number of well-documented alterations in immune functions such as complement deficiency and impaired dendritic cell maturation (1995, Shultz et al., J Immunol 154:180-191).
Recently, several approaches have been used to improve human cell engraftment and the unbalanced lineage differentiation in CD34+ cell engrafted mice. These include transient approaches such as hydrodynamic injection of plasmid DNA (2009, Chen et al., Proc Natl Acad Sci USA 106:21783-21788), injections of cytokines, and infections of mice or CD34+ cells with lentiviruses (2010, O'Connell et al., PLoS ONE 5:e12009; 2009, Huntington et al., J Exp Med 206:25-34; 2009, van Lent et al., J Immunol 183:7645-7655.). Alternatively, transgenic expression of human MHC molecules has been demonstrated to improve the development of antigen-specific immune responses in vivo (2009, Jaiswal et al., PLoS ONE 4:e7251; 2009, Strowig et al., J Exp Med 206:1423-1434; 2011, Danner et al., PLoS ONE 6:e19826). Nonetheless, overexpression of cytokines might also have detrimental side effects due to the unphysiological expression such as in mice transgenic for GM-CSF, and IL-3 (2004, Nicolini et al., Leukemia 18:341-347). An alternative approach to provide human growth factors in vivo is to genetically engineer mice and replace the mouse genes with their human counterparts resulting in their expression in the appropriate niche at physiological levels. Indeed, faithful replacement of mouse GM-CSF and IL-3 as well as thrombopoietin (TPO) group has resulted in improved development of human macrophages in the lung and HSPC and HPC maintenance in the bone marrow, respectively (2011, Rongvaux et al., Proc Natl Acad Sci USA 94:5320-5325; 2011, Willinger et al., Proc Natl Acad Sci USA 108:2390-2395). Notably, in human TPO knockin mice, despite a highly increased engraftment level of stem and progenitor cells in the bone marrow, no changes were observed in the periphery, demonstrating the existence of limiting factors in the periphery such as destruction by phagocytes.
One application of mice with functional human immune systems is the development and testing of human vaccines. Historically, the induction of immune responses in vivo has been relatively inefficient (2004, Traggiai et al., Science 304:104-107; 2002, Ito et al., Blood 100:3175-3182; 2005, Ishikawa et al., Blood 106:1565-1573; 2005, Shultz et al., J Immunol 174:6477-6489; 2006, Baenziger et al., Proc Natl Acad Sci USA 103:15951-15956). Several studies have reported successful pathogen-specific immune responses upon infection. Although it was reported that around 50% of mice produced virus-specific IgM and IgG upon dengue virus infection (2007, Kuruvilla et al. Virology 369:143-152), other studies reported frequencies below 20% of mice producing antigen-specific IgM and IgG after HIV and EBV infection (2006, Baenziger et al., Proc Natl Acad Sci USA 103:15951-15956; 2008, Yajima et al., J Infect Dis 198:673-682). Upon immunization with adjuvant and antigen, class switching of antigen-specific immunoglobulins is also historically inefficient with only a fraction of immunized animals showing antigen specific IgG responses (2004, Traggiai et al., Science 304:104-107; 2002, Ito et al., Blood 100:3175-3182; 2005, Ishikawa et al., Blood 106:1565-1573; 2005, Shultz et al., J Immunol 174:6477-6489; 2009, Watanabe et al., Int Immunol 21:843-858; 2010, Becker et al., PLoS ONE 5). These studies included NSG and BALB/c DKO mice and different adjuvant/antigen combinations.
There is a need in the art for non-human animals able to support and sustain engraftment with a human hematopoietic system. The present invention addresses this unmet need in the art.